The goal of this project is to better understand activation of transcription initiation by eukaryotic RNA polymerase II (RNApII), a process that is often abnormal in cancer cells. The experiments proposed will combine Colocalization Single-Molecule Spectroscopy (CoSMoS, a TIRF microscopy technique for simultaneously analyzing hundreds of single-molecule events) with Saccharomyces cerevisiae nuclear extracts that support robust transcription activation. Extracts will be prepared from strains expressing two or three transcription factors each fluorescently labeled with a different color. These extracts will be combined with a transcription activator (Gal4-vp16 or Gcn4) labeled with yet another color, and DNA templates immobilized on the microscope slide. CoSMoS allows precise measurements of interaction dynamics between promoter DNA, activators, co-activators, and the RNApII transcription machinery. Specific Aim 1 will measure temporal relationships between activator, RNApII, and the co-activator Mediator. These results will show whether Mediator and RNApII arrive at and leave promoters as a complex, or whether Mediator can stay bound to support multiple RNApII binding events. Similarly, they will reveal whether activator recruitment of Mediator involves cooperative thermodynamic interactions, or instead if activator kinetically accelerates formation of a Mediator-PIC complex that no longer requires bound activator. Specific Aim 2 is a similar analysis of how activator affects promoter binding of the coactivators Swi/Snf, SAGA, and NuA4. These three factors act upon nucleosomes, so comparative experiments will be carried out on naked versus chromatinized templates. Experiments labeling different combinations of coactivators will reveal if their binding is independent, sequential, simultaneous, or mutually exclusive. Finally, Specific Aim 3 will compare effects of having single versus multiple activators bound at the promoter. One set of experiments will monitor matched promoters having single versus multiple Gal4 binding sites. Gal4-vp16 will also be compared to Gal4-vp64, an even stronger activator that carries four tandem vp16 activation domains fused to Gal4 DNA binding domain. These experiments will reveal whether transcriptional synergy reflects increased binding frequencies, durations, and/or co-occupancy of coactivators and Mediator/RNApII. Together, these single molecule experiments will reveal fundamental information about transcription activation that has been impossible to glean from ensemble biochemical or genomic techniques. The yeast system is well established as an excellent model system for all eukaryotes, and findings here will provide deeper understanding of the mammalian homologs that are very frequently mutated in cancer.